Quarterly Reviews of Biophysics 40, 2 (2007), pp. 163–189. f 2007 Cambridge University Press 163 doi:10.1017/S0033583507004647 Printed in the United Kingdom Quantitative studies of ribosome conformational dynamics Christopher S. Fraser and Jennifer A. Doudna* Department of Molecular and Cell Biology & Department of Chemistry, Howard Hughes Medical Institute, University of California at Berkeley, Berkeley, CA, USA Abstract. The ribosome is a dynamic machine that undergoes many conformational rearrangements during the initiation of protein synthesis. Significant differences exist between the process of protein synthesis initiation in eubacteria and eukaryotes. In particular, the initiation of eukaryotic protein synthesis requires roughly an order of magnitude more initiation factors to promote efficient mRNA recruitment and ribosomal recognition of the start codon than are needed for eubacterial initiation. The mechanisms by which these initiation factors promote ribosome conformational changes during stages of initiation have been studied using cross-linking, footprinting, site-directed probing, cryo-electron microscopy, X-ray crystallography, fluorescence spectroscopy and single-molecule techniques. Here, we review how the results of these different approaches have begun to converge to yield a detailed molecular understanding of the dynamic motions that the eukaryotic ribosome cycles through during the initiation of protein synthesis. 1. Introduction 164 2. Protein synthesis and ribosome structure 164 3. Overview of the initiation mechanism and factors 165 4. Initiation-factor binding sites on the small ribosomal subunit 169 4.1 Chemical cross-linking approaches 169 4.2 Footprinting 170 4.3 Site-directed structural probing 174 4.4 Cryo-EM and X-ray crystallographic studies 176 4.5 Fluorescence techniques 179 4.6 Single-molecule analysis 181 5. Conclusions and future perspectives 182 6. Acknowledgements 183 7. References 183 * Author for correspondence: Dr J. A. Doudna, Department of Molecular and Cell Biology & Department of Chemistry, Howard Hughes Medical Institute, University of California at Berkeley, Berkeley, CA, USA. Tel.: (510) 643-0225; Fax: (510) 643-0080; Email: [email protected] 164 C. S. Fraser and J. A. Doudna 1. Introduction Over the past few years our structural understanding of the ribosome and the initiation factors that promote formation of competent ribosomes on mRNA has increased dramatically. Although detailed analysis of eukaryotic protein synthesis has lagged behind that of bacterial systems, we are beginning to understand the underlying mechanisms of eukaryotic translation initiation despite the current lack of molecular structures of eukaryotic ribosomes. The goal of this review is to summarize the biophysical approaches and resulting discoveries that have revealed substantial information about ribosome dynamics during the initiation of eukaryotic protein synthesis. We also discuss the challenges that lie ahead in understanding the functional states and dynamic motions of the ribosome during the initiation process. While it has been known for some time that multiple initiation factors are required for this process, our molecular understanding of how these factors promote each stage of initiation has not been clear. We have tried to present what we see as important developments in our understanding of this mechanism and direct the reader to recent specialized reviews for greater detail about specific events and the initiation factors that appear to promote them. 2. Protein synthesis and ribosome structure Protein synthesis in all organisms is carried out by ribosomes, which consist of two ribonucleo- protein subunits that translate mRNA into protein by catalyzing the formation of peptide bonds. There are four stages to this process: initiation, elongation, termination, and recycling. During initiation, the two ribosomal subunits associate at the initiation codon of the mRNA, which is recognized by virtue of a methionyl initiator tRNA bound to the peptidyl (P) site of the small ribosomal subunit. Elongation requires the ribosome to decode the mRNA sequence by repeated cycles of three distinct steps: (1) recruitment of aminoacyl tRNAs to the aminoacyl (A) site of the small ribosomal subunit, (2) formation of a peptide bond between the incoming amino acid and the amino acid on the tRNA in the P-site, and (3) translocation of the mRNA and tRNAs so that the next codon is placed in the A-site of the ribosome. Protein synthesis terminates when a stop codon is placed in the A-site, causing the finished peptide to be released from the ribosome. Finally, ribosome recycling involves dissociation from the mRNA and the ejection of the bound deacylated tRNA so that the ribosome can enter another round of protein synthesis. Each stage of this process requires the ribosome to undergo many dynamic motions so that the mRNA is translated accurately and efficiently into protein (for details of the stages of eukaryotic protein synthesis see Kapp & Lorsch, 2004; Marintchev & Wagner, 2004; Hinnebusch et al. 2007; Pestova et al. 2007). Ribosomes from the three kingdoms of life possess many sequence and structural similarities, indicating a common evolutionary origin. Bacterial ribosomes consist of a small (30S) subunit and a large (50S) subunit that together form the 70S ribosome. The small subunit comprises a single 1540-nt RNA (16S) and 21 proteins, while the large subunit includes a small 120-nt RNA (5S) along with a 2900-nt RNA (23S) and some 33 proteins. While eukaryotic ribosomes are larger in size than their cousins, they still possess a small (40S) subunit and a large (60S) subunit that form an 80S ribosome. The small subunit consists of a 1900-nt RNA (18S) and 33 proteins, while the large subunit comprises a 12-nt RNA (5S), a 160-nt RNA (5Á8S), a 4700-nt RNA (28S) and 49 proteins. Many years of biochemical work and more recent X-ray and cryo-electron micro- scopic (cryo-EM) structures have helped us to understand more about the processes by which Quantitative studies of ribosome conformational dynamics 165 ribosomes function and are regulated during each stage of protein synthesis. Structural studies suggest that although different in size, ribosomes from all three kingdoms of life share a high degree of common structure, which likely reflects conservation in the fundamental process of protein biosynthesis (Taylor et al. 2007). Recent X-ray crystallographic structures of the ribosome and its subunits from bacteria and archaea have indicated that the interactions between mRNA, tRNA and rRNA required for elongation primarily involve RNA–RNA contacts, and it is anti- cipated that this will also be true for eukaryotic ribosomes as higher-resolution structures be- come available (Noller, 2007). For all ribosomes, the small subunit has been identified as that which contacts and decodes the mRNA, whereas the large subunit contains the catalytic center that promotes peptide bond formation. The role of extra rRNA sequences in eukaryotes, called expansion segments, along with the extra proteins that they bind is currently unclear. These expansion regions are found predominantly around the periphery of the small subunit, as de- termined by comparing cryo-EM structures (reviewed in Taylor et al. 2007). There are functional differences in a number of stages of protein synthesis between the kingdoms of life, particularly during the initiation of protein synthesis, which likely account for some of these eukaryotic- specific regions of ribosomes. In addition, the differences in how the ribosome functions in protein transport and folding, as well as the fact that eukaryotic ribosomes must also pass through the nuclear pore complex, may also reflect the requirement of ribosome features that are specific to eukaryotes. 3. Overview of the initiation mechanism and factors A key regulatory step in protein synthesis is the mechanism by which ribosomes initiate protein synthesis. Some similarities and differences in this mechanism are immediately apparent between bacteria and eukaryotes and have been the subject of recent reviews (Kapp & Lorsch, 2004; Marintchev & Wagner, 2004; Merrick, 2004; Jackson, 2005; Kozak, 2005). A model of the steps involved is presented in Fig. 1. In each case, the ribosome must first separate into its two subunits, with the small subunit engaging a mRNA and selecting the start site for translation. Binding of a dedicated Met-tRNA to the small subunit in a GTP-dependent manner to recognize the initiation codon is also conserved, although the regulation of this step is significantly different in eukaryotes (Hinnebusch et al. 2007; Proud, 2006). In bacteria, the Shine–Dalgarno (SD) se- quence of the mRNA base pairs with a complementary sequence in the 16S rRNA, enabling 30S binding and initiation on polycistronic mRNAs directly at the site of the AUG codon (Shine & Dalgarno, 1974; Steitz & Jakes, 1975). Importantly, the sequence located near the 3k end of the 16S rRNA that recognizes the SD sequence is specifically deleted in the eukaryotic 18S rRNA, thereby preventing direct recognition of mRNA (Hagenbuchle et al. 1978). Instead, the majority of eukaryotic mRNAs initiate by a scanning mechanism that involves 40S subunit loading at the 5k end of the mRNA and subsequent migration to the AUG codon (Kozak, 1989b). The regu- lation of initiation codon recognition also appears to differ between bacteria and eukaryotes. Initiation in bacteria is very sensitive to the degree of secondary structure at the initiation site, while eukaryotes